DESCRIPTION: The evolution of novel targeted- and chemo-therapies for LC has achieved modest improvement in median survival for advanced LC, but they offer no clear path to treatments that could make this a chronic, rather than fatal disease. Furthermore, for the majority of the more than 220,000 new lung cancer cases diagnosed annually in the US for which most targeted therapy is not an option, durable responses with chemotherapy are uncommon and median survival after relapse is only 6.5 months. This presents a daunting challenge to develop a different outside of the box approach with low systemic toxicity that in turn will allow chronic dosing to achieve and sustain therapeutic responses. The Cancer Genome Atlas has interrogated over 600 non-small cell lung cancers (NSCLCs) and revealed that virtually all tumors contain hundreds of genes that have densely cytosine methylated promoter regions leading to reduced transcription. Epigenetic therapy through its ability to awaken these genes offers a strategy that could ultimately produce durable and sustained tumor regression. A Phase II trial combining non-cytotoxic doses of the demethylating agent Vidaza (5- azacytidine) with the histone deacetylase (HDAC) inhibitor entinostat (MS275) showed promising results in patients with refractory, advanced NSCLC. The required continuous daily subcutaneous dosing schedule may constrain the expansion of epigenetic therapy in Phase III trials or in adjuvant therapy, while oral formulations are problematic due to hydrolysis; both dosing routes are subjected to catabolism by cytidine deaminase that greatly reduces Vidaza half-life. These barriers to expanding use and improving the response to epigenetic therapy could be mitigated by aerosolized administration that would deliver Vidaza directly to the lungs. The absorption of the aerosol into the pulmonary vasculature also avoids hepatic first pass, thus eliminating initial inactivation by cytidine deamination, while providing systemic dose to treat occult metastases. The orthotopic lung cancer model we developed in which xenografts of human lung cancer-derived cell lines are engrafted throughout the lungs of the nude rat allows testing of combination therapies and different routes of administration. Our first studies demonstrated that systemic delivery of Vidaza and entinostat at doses and schedule similar to the Phase II clinical trial were synergistic in suppressing tumor growth by 60% and induced reprogramming of the epigenome as detected by gene demethylation and re-expression. Recent studies now show that an aqueous formulation of Vidaza administered as an aerosol can effectively reduce lung tumor burden and induce common global demethylation of 300 genes at one-third the comparable effective systemic dose. The major goals of this application are to develop an optimal aerosol formulation of Vidaza, and to develop dosing strategies in combination with entinostat and inhibitors of histone methylation that maximally affect tumor burden and reprogramming of the epigenome in NSCLC using our orthotopic model. The culmination of this work will enable future innovative Phase I/II lung cancer clinical trials.